This invention relates to a photometric apparatus for spectroscopic analysis of small samples in solution. The improved apparatus includes a flow cell, uniquely suitable for aqueous solutions encountered in liquid chromatography or capillary electrophoresis, has high optical throughput, a long path length and a small cross-section.
It is well known that low concentrations of analyte in solution can be detected spectroscopically with greater sensitivity when the optical path length through the sample is long. When, as is frequently the case, the quantity of sample is limited, the optimum cell has a small cross-section. However, it is also necessary to pass sufficient optical power through the sample to maintain a good signal-to-noise ratio. The first two requirements can be met when the sample solution flows through a capillary tube of small cross-section and the optical path is along the tube axis. Large optical throughput can be achieved when the light is guided along the capillary similar to the way light is guided along an optical fiber.
Two possibilities exist for guiding light along a capillary. First, by total internal reflection at the boundary between the liquid and the capillary wall, and second, by total internal reflection between the outside diameter of the capillary and the surrounding air. In either case the tubing material must be transparent over the wavelength range of interest. The first approach is preferred, since light is confined to the liquid sample, but there is a serious limitation. The refractive index of the liquid sample must be significantly above that of the tube material, otherwise the light will pass out of the liquid into the tubing wall. Stone, in U.S. Pat. No. 3,814,497 using high-index chlorinated organic liquids and silica tubes demonstrated efficient optical transmission in the visible and near infrared wavelengths and shows a sensitive cell for Raman spectroscopy, in U.S. Pat. No. 3,770,350. X.Xi and E.S. Yeung, Anal. Chem. 1990, 62, page 1580, describes sensitive absorbance detection in capillary liquid chromatography using silica tubes and a high-index mobile phase.
Most high performance liquid chromatography (HPLC), capillary LC and capillary electrophoresis (CE) separations are done in aqueous media. The refractive index of water over the visible and ultraviolet (UV) spectral range is much less than that of fused silica which is the only existing capillary tube material to combine good sample compatibility with optical transparency to wavelengths below 200 nm. For example, at the sodium D line, 589 nm, the indices of water and fused silica are 1.333 and 1.458. Both indices rise towards the UV. At 254 nm they are 1.374 and 1.505. Thus for the majority of liquid chromatographic or CE applications, light can not be guided in the liquid along a fused silica capillary tube to achieve the desired level of sensitivity.
The index of conventional fluoropolymer (Teflon) materials, while still higher than water, is closer to it than is fused silica. For example, the index of Teflon PFA in the visible is 1.34 to 1.35. Teflon fluoropolymer tubes (FEP, PFA or PTFE) have been used with higher-index aqueous salt solutions to transmit power, as shown by Nath, U.S. Pat. No. 4,009,382, and as a colorimeter cell, as shown by Uffenheimer, U.S. Pat. No. 3,954,341. Adding solutes to elevate the refractive index would be an undesirable requirement for many chromatographic or CE separations. The use of conventional Teflon materials for an axially-illuminated detector cell has a more serious drawback--poor transparency at shorter UV wavelengths where its partial crystallinity scatters light. UV measurements down to 200 nm and below are not possible.
The second approach to guiding light along a capillary tube overcomes the refractive index and UV transparency limitations discussed above. Long capillary cells have been constructed of fused silica, where light guiding occurs at the boundary between the silica tube outside diameter and air. Because of the large refractive index difference between silica and air, this is a very efficient light guide of large numerical aperture, as long as the outside diamter of the silica is clean and smooth. Unfortunately, the light spends much of its time in the capillary walls, making periodic passes through the sample in the lumen. Thus, only a fraction of the optical path length is effective for analytical purposes. When light is guided at the tube outside diameter/air interface, much of the light which can be guided follows helical paths which never come into contact with the sample. This light merely adds noise to the measurement, further degrading the detector's sensitivity. An example of light guiding at the outer wall/air interface of a transparent tube is disclosed by Carlson in U.S. Pat. No. 4,477,186.
Thus, all presently available long path cells for spectroscopic detection of low concentrations and small quantities of an analyte in aqueous solution have major drawbacks and limitations. Accordingly, it would be desirable to provide an improved liquid flow cell of small cross-section and long path where light is guided axially along its length and confined to the liquid sample. Such a cell should perform well with small quantities of solute in water or aqueous solvents common in reverse phase HPLC, capillary LC or CE.